Brevibacillus agri, preparation thereof, method for preparing surfactant and use thereof

ABSTRACT

The present disclosure relates to the technical field of application of bioengineering technology in microbial oil recovery, and discloses a Brevibacillus agri strain and preparation thereof and a method for preparing surfactant, and use thereof. The Brevibacillus agri strain is deposited in the China General Microbiological Culture Collection Center under the accession number CGMCC No. 9983. The Brevibacillus agri and its preparation may effectively enhance the crude oil recovery; the method for preparing the surfactant allow the lipopeptide biosurfactant to have good physical properties, effectively reduce the surface tension, and have good emulsifying performance for petroleum, various hydrocarbons and lipids.

CROSS REFERENCE TO RELATED APPLICATION

This application claims the priority of Chinese Patent Application No.202010813094.X, entitled “Brevibacillus agri, preparation thereof andmethod for preparing surfactant and use thereof” filed with the ChinaNational Intellectual Property Administration on Aug. 13, 2020, which isincorporated herein by reference in its entirety.

TECHNICAL FIELD

The disclosure relates to the application of bioengineering technologyin the field of microbial enhanced oil recovery, and in particular to aBrevibacillus agri strain and preparation thereof, and to a method forpreparing surfactant and use of the surfactant.

BACKGROUND ART

Microbial enhanced oil recovery (MEOR) is considered as the replacementtechnology for tertiary oil recovery (i.e. the quaternary recovery) dueto its simple operation, economy, environmental protection andsustainable development characteristics. In early 2017, microbialenhanced oil recovery was rated as one of the top ten world petroleumtechnology advances by PetroChina in 2016, which is becoming more andmore widely used in China's oilfields, from the oilfields of easternChina, for example Daqing, Shengli, Dagang, Huabei and Jilin, to theoilfields of western China, for example Xinjiang, Qinghai, Changqing andTuha. In particular, in recent years, the microbial enhanced oilrecovery has been successfully applied in low permeability reservoirsand heavy oil reservoirs. The indigenous microorganisms of the oilreservoir can metabolize different substrates, and producebiosurfactants, biopolysaccharides, biogas, bioacids, biosolvents, etc.,and synergistically enhance oil recovery. The viscosity reduction ofcrude oil through dispersion and emulsification by the producedbiosurfactant is an important mechanism of MEOR.

For example, the Chinese patent with application number CN 01115920.0discloses Bacillus brevis and its use in removing sulfur from thesulfur-containing organic compounds. The Bacillus brevis involved in thedisclosure is Bacillus brevis R-6 strain which was deposited in “ChinaGeneral Microbiological Culture Collection Center” on Apr. 29, 2001under the accession number of CGMCC NO. 0571. R-6 strain was isolatedfrom aerobic activated sludge in oily wastewater treatment pool of arefinery, from oil contaminated soil around oil wells and from soilsamples around coal mines with high sulfur content. The Bacillus brevisR-6 strain and its non-growth stationary cell liquid, immobilized cell,cell liquid culture or crude enzyme extract can remove sulfur fromsulfur-containing organic compounds by oxidative cleavage of C-S bond.

For another example, the Chinese patent with application number CN01115920.0 discloses a method for controlling the process of microbialdegradation of crude oil, including determining the type, concentration,dosage and other parameters of surfactant by studying the effect ofsurfactant on microbial growth and crude oil degradation. The disclosurealso provides a degradation agent for petroleum pollutants, includingoil-producing bacteria and identified surfactant. CN 01115920.0 alsoprovides a method for using microorganisms to degrade petroleumpollutants, including injecting an identified petroleum pollutantdegradation agent into the polluted environment. CN 01115920.0 allows tocontrol the process of microbial degradation of crude oil, and reduce oreliminate the oil pollution in the environment through sharing of thesurfactant and degradation bacteria.

However, there is no report about use of Brevibacillus Agri. and itspreparation, and its metabolic organism in enhancing oil recovery.

In view of the technical gap existing in the prior art, the presentdisclosure provides a Brevibacillus agri, its use and a method forpreparing surfactant.

SUMMARY OF THE INVENTION

The present disclosure provides a Brevibacillus agri, its use and methodfor preparing surfactant.

The present disclosure provides the following technical schemes.

A Brevibacillus agri strain, wherein the accession number of theBrevibacillus agri strain is CGMCC No. 9983.

In one aspect, the present disclosure provides a Brevibacillus agripreparation, wherein the Brevibacillus agri preparation comprises theBrevibacillus agri strain with accession number CGMCC No. 9983, whereinthe Brevibacillus agri strain is a liquid bacterial preparation.

In another aspect, the present disclosure provides a method forpreparing biosurfactant, wherein the method comprises fermenting theBrevibacillus agri or the Brevibacillus agri preparation in a nutrientmedium to produce a lipopeptide biosurfactant.

In one embodiment, the nutrient medium comprises: MgSO₄ 0.2 g/L, K₂HPO₄1.0 g/L, KH₂PO₄ 1.0 g/L, Na₂HPO₄ 4.0 g/L, NaCl 10.0 g/L, NaNO3 10.0 g/L,crude oil 10.0 g/L and molasses 2-3 g/L, pH is 7.0, and fermentationtemperature is 25-60° C.

In one embodiment, the nutrient medium comprises 1-5 mL of solution oftrace elements and 1-10 mL of solution of vitamin complex.

In one embodiment, the method further comprises removing bacteria afterfermentation.

In one embodiment, the method for fermenting the Brevibacillus agri orthe Brevibacillus agri preparation in a nutrient medium comprises thefollowing steps:

-   -   1.1 inoculating the Brevibacillus agri strain preserved on slant        culture medium on the culture plate through streak inoculation        by inoculating loop, culturing at 30° C. for 20 h to activating;    -   1.2 inoculating the Brevibacillus agri strain from the culture        plate into the primary seed shaker by picking three loops of        bacteria, and culturing at 30° C. and 180 r/min for 18 h;    -   1.3 inoculating the primary seed liquid into the second stage        shaker according to 2% of the volume of the primary seed shaker        and culturing under the same conditions as in step 1.2 for 18 h;    -   1.4 inoculating the second stage liquid into the fermentation        shaker according to 2% of the volume of the second stage shaker        and culturing under the same conditions until the spore rate        reaches 100% to obtain fermentation broth.

In one embodiment, the method further comprises a method for extractingthe biosurfactant, the method for extracting the biosurfactant comprisesthe following steps:

-   -   2.1 adjusting the pH of the fermentation broth to 8, removing        the bacteria by centrifuging twice at 4° C. and 9000 r/min for        20 min, adjusting the pH of supernatant to 2.0 with 12 mol/L        hydrochloric acid, observing flocculent precipitates, allowing        the supernatant to stand at 4° C. overnight;    -   2.2 centrifuging the fermentation broth at 4° C. and 9000 r/min        for 30 min, pouring supernatant out, washing the precipitate in        the centrifuge tube with hydrochloric acid solution with pH of        2.0, adjusting the pH of precipitate to 7.0 with 1 mol/L NaOH,        and freeze drying to obtain yellowish brown loose solid to        prepare crude product of surfactant;    -   2.3 packing the crude product of surfactant with an aluminum        foil, putting into a Soxhlet extractor, extracting the crude        product of surfactant with 150 mL dichloromethane for 10 h,        controlling the dripping rate of organic solvent at 1-2 drops        per second, removing the solvent by rotary evaporation after        extraction, washing the alkanes out with 3 times volume of        n-hexane to obtain brownish yellow precipitate, and freeze        drying to prepare the purified biosurfactant.

In yet another aspect, the present disclosure provides a biosurfactant.

In yet still another aspect, the present disclosure provides a methodfor preparing lipopeptide biosurfactant, wherein the lipopeptidebiosurfactant is metabolized by the Brevibacillus agri strain.

In yet still another aspect, the present disclosure provides a use ofthe Brevibacillus agri strain in enhancing oil recovery.

In yet still another aspect, the present disclosure provides a use ofthe Brevibacillus agri preparation in enhancing oil recovery.

In yet still another aspect, the present disclosure provides a use ofthe biosurfactant in enhancing oil recovery.

Compared with the prior art, the present disclosure has the followingbeneficial effects:

-   -   1. The lipopeptide biosurfactant of the present disclosure has        good physical properties, may effectively reduce the surface        tension, and has good emulsification for petroleum, various        hydrocarbons and lipids;    -   2. The Brevibacillus agri and its preparation may effectively        enhance the crude oil recovery.

Depository information

Depository address: Institute of Microbiology, Chinese Academy ofSciences, No.3, No.1 courtyard, Beichen West Road, Chaoyang District,Beijing, China;

Depository date: Nov. 18, 2014;

Depository name: Brevibacillus Agri;

Depository institution: China General Microbiological Culture CollectionCenter(CGMCC);

Accession number: CGMCC No. 9983.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the schematic diagram of the colony of B5-2 strain inExample 1 of the present disclosure;

FIG. 2 shows the schematic diagram of Gram staining microscopeobservation of B5-2 strain in Example 1 of the disclosure;

FIG. 3 shows the transmission electron microscope of B5-2 strain inExample 1 of the present disclosure;

FIG. 4 shows the schematic diagram of the effect of different carbonsources on the surface tension in Example 3 of the present disclosure;

FIG. 5 shows the schematic diagram of the effect of different nitrogensources on the surface tension in Example 3 of the present disclosure;

FIG. 6 shows the schematic diagram of emulsifying property test ofsurfactant produced by B5-2 strain in Example 4 of the disclosure;

FIG. 7 shows the schematic diagram of changes in the mass fraction ofcrude oil components before and after the action of B5-2 strain inExample 5;

FIG. 8 shows the schematic diagram of the chromatographic-massspectrometry(GC/MS) analysis of the content of saturated hydrocarboncomponents before the action of B5-2 strain in Example 5 of the presentdisclosure;

FIG. 9 shows the schematic diagram of the chromatography-massspectrometry(GC/MS) analysis of the content of saturated hydrocarboncomponents after the action of B5-2 strain in Example 5 of the presentdisclosure;

FIG. 10 shows the chromatography-mass spectrometry(GC/MS) spectrum ofaromatic hydrocarbon of TH191 crude oil in Example 5 of the disclosure;

FIG. 11 shows the GC/MS spectrum of aromatic hydrocarbons after theaction of B5-2 strain in TH191 crude oil in Example 5 of the disclosure;

FIG. 12 shows the schematic diagram of the relative abundance of basicheteroatoms in crude oil TH191 before degradation in Example 5 of thepresent disclosure;

FIG. 13 shows the relative abundance of basic heteroatoms in crude oilTH191-S-4-OK after degradation in Example 5 of the present disclosure.

DETAILED DESCRIPTION OF THE EMBODIMENTS

The present disclosure is further described in combination with specificexamples, and the characteristics and technical effects in applicationof Brevibacillus Agri of the present disclosure are described. Thefollowing examples are not intended to limit the present disclosure, butonly to illustrate the present disclosure. Unless otherwise specified,the experimental methods used in the following examples will usuallyfollow conventional conditions unless specific conditions are specifiedin the examples. Unless otherwise specified, the materials and reagentsused in the following examples can be obtained from commercial sources.

The present disclosure provides a Brevibacillus Agri strain isolatedfrom the extreme environment of oil reservoir. The strain has goodcharacteristics of degrading crude oil. The Brevibacillus Agri strainand its preparation may degrade macromolecular hydrocarbons and polarcompounds of crude oil, change the hydrophilic and lipophilic propertiesof water interfacial active substances of crude oil, reduce viscosityand enhance crude oil recovery. The present disclosure provides for thefirst time a method for preparing lipopeptide biosurfactant byfermentation of Brevibacillus Agri and a method for extractingmetabolites of Brevibacillus Agri. The present disclosure also providesa Brevibacillus agri preparation and its use in enhancing oil recovery.

Example 1: Isolation, Identification and Deposition ofBiosurfactant-Producing Strains

1. Enrichment Culture and Isolation of Biosurfactant-Producing Strains

According to the conventional screening method, 10 mL of oil-watersample collected from oilfields of western China was put into 100 mL ofsterilized crude oil culture medium (2% crude oil, V: V) and cultured at30° C. and 180 rpm for 96 h. Then 5% of the enrichment culture solutionof the experimental group with high emulsification and dispersion degreeof crude oil was transferred to fresh culture medium and cultured at 30°C. and 180 rpm for 96 h. After repeated enrichment and cultivation for10 rounds, the experimental group with the best emulsification anddispersion of crude oil was selected. 100 μL fermentation broth wasspread on LB-agar-medium and cultured at 30° C. for 48 h; singlecolonies with different morphology were cultured on LB-agar-medium andpurified by a streak plate method and cultured at 30° C. for 48 h. Afterenrichment culture, single colony was inoculated into the crude oilinorganic salt culture medium with crude oil as carbon source, andcultured at 30° C. and 180 rpm for 96 h. The degree of emulsificationand dispersion of crude oil and the surface tension of fermentationbroth were observed, the surface tension of the fermentation liquide wasmeasured and the strain with the largest decrease in surface tension wasselected.

In this example, through preliminary screening, it is found that thereare microorganisms that can emulsify and disperse the crude oil well andreduce the surface tension of the culture medium in the sample. Afterthe cultivation and domestication, a biosurfactant-producing strain isisolated and named. The diameter of oil drainage circle is 6.65 cm, andthe surface tension of fermentation broth is reduced to 30.68 mn/m. Themetabolites of B5-2 strain have excellent surface activity and can beused in microorganisms to enhance crude oil recovery.

TABLE 1 Determination of surfactant-producing microorganisms OptimumSurface tension Diameter of oil drainage Strains temperature/° C. mN/mcircle/cm B5-2 30 30.68 6.65

2. The physiological and biochemical identification and preservation ofB5-2 strain are based on the “Manual Of Identification In CommonBacterial”. The B5-2 strain is identified from the aspects of individualmorphological characteristics, colony characteristics, stainingreaction, physiological and biochemical reaction, and the genus name ofB5-2 strain is identified. The identification includes morphologicalobservation, Gram staining, catalase reaction, oxidase test, glucoseoxidation fermentation test, methyl red test, etc. The results are shownin Table 2.

TABLE 2 identification results of physiological and biochemicalcharacteristics of B5-2 strain Physiological and biochemicalcharacteristics L1 strain Glucose + Maltose + Lactose − Galactose −Unique carbon Rhamnose − source test Raffinose − Sorbitol − Sucrose +Ammonium chloride + Unique Sodium nitrate + nitrogen source Sodiumnitrite + test Indole test + Hydrogen sulfide production test − Methylred test − Acetylmethylcarbinol test + Starch hydrolysis test + Citrateutilization test + Temperature range for growth 25° C.-60° C. Salinityrange for growth >7%

The colony is round, having convex surface, milky white, smooth andmoist surface. B5-2 strain is rod-shaped and produces spores, and thespores has expanded terminal. The micrograph is shown in FIG. 1 and FIG.2 . The electron micrograph of B5-2 strain made in the Institute ofmicrobiology, Chinese Academy of Sciences is shown in FIG. 3 . Theelectron micrograph of JEM-1400 shows that the B5-2 strain has capsuleand surrounded periflagella.

According to the conventional strain identification method, the genomicDNA of B5-2 strain was extracted, and primers were designed for PCRamplification. The amplified products were detected by agarose gelelectrophoresis, and DNA sequencing was performed by Sangon. It wasdetermined that the B5-2 strain is Brevibacillus Agri. strain (thesimilarity was 99%) through the search and homology comparison of thePCR product sequence of B 5-2 in NCBI BLAST.

The B5-2 strain of this example can be preserved by using the followingmethod:

(1) Short term preservation: the above-mentioned strains are inoculatedon slant culture medium through streak inoculation, cultured at 35° C.for 48 h, and stored at 4° C.

(2) Long term preservation: a glycerol cryopreservation method may beused. The strains are inoculated from the fresh slant culture mediuminto the tube with 1.5 mL 30% sterilized glycerin by inoculating loopfor cryopreservation at −80° C. Alternatively, a skimmed milkcryopreservation method may be used. The strains are inoculated from thefresh slant culture medium into the tube with sterilized skimmed milkfor cryopreservation at −80° C.

Brevibacillus Agri B5-2 strain was deposited in China GeneralMicrobiological Culture Collection Center (Institute of Microbiology,Chinese Academy of Sciences, No.3, No.1 courtyard, Beichen West Road,Chaoyang District, Beijing, China;) on Nov. 18, 2014, with the accessionnumber of CGMCC No. 9983.

Example 2: Extraction of B5-2 Strain Metabolites

1. Fermentation of B5-2 Strain

The fermentation culture medium of Brevibacillus Agri B5-2 strainincluded: MgSO₄ 0.2g/L, K₂HPO₄ 1.0 g/L, KH₂PO₄ 1.0 g/L, Na₂HPO4 4.0 g/L,NaCl 10.0 g/L, NaNO₃ 10.0 g/L, crude oil (heavy oil) 10.0 g/L andmolasses 2-3 g/L, pH 7.0; appropriate amount of solution of traceelements and vitamins complex, pH 6-8. The culture medium was sterilizedat 115° C. for 30 min. The fermentation temperature was 30° C.

The strain preserved on slant culture medium on the culture plate wasinoculated through streak inoculation with a inoculating loop, activatedand cultured at 30° C. for 20 h. Then three loops of the strain waspicked from the culture plate into the primary seed shaker (250 mLtriangular flask, loading volume of liquid was 50 mL), and cultured at30° C. and 180 r/min for 18 h (each inoculation loop contained more than3 single colonies with obvious characteristics). The primary seed liquidwas inoculated into the second stage shaker (250 mL triangular flask,the loading volume of liquid is 100 mL) in 2% of the volume of theprimary seed shaker and cultured under the same conditions as in step1.2 for 18 h. The second stage liquid was inoculated into thefermentation shaker (500 mL triangular flask, the volume of liquid is150 mL) in 2% of the volume of the second stage shaker and culturedunder the same conditions until the spore rate reaches 100% to obtainfermentation broth.

2. Extraction and Identification of Lipopeptide Produced By B5-2

The pH of fermentation broth was adjusted to 8, and the bacteria wasremoved by centrifuging twice at 4° C. and 9000 r/min for 20 min. The pHof supernatant was adjusted to 2.0 with 12 mol/L hydrochloric acid,flocculen precipitates were observed, and the supernatant was kept at 4°C. overnight; the fermentation broth was centrifuged at 4° C. and 9000r/min for 30 min, the supernatant was poured out. The precipitate waswashed in the centrifuge tube with hydrochloric acid solution with pH of2.0. The pH of precipitate was adjusted to 7.0 with 1 mol/L NaOH, andfreeze dried to obtain a yellowish brown loose solid to prepare crudeproduct of surfactant. The crude product of surfactant was packed withan aluminum foil, and put into Soxhlet extractor. The crude product ofsurfactant was extracted with 150 mL dichloromethane for 10 h. Thedripping rate of organic solvent was controlled at 1-2 drops per second.The solvent was removed by rotary evaporation after extraction. Thealkanes were washed off with 3 folds of volume of n-hexane to obtainbrownish yellow precipitate, and freeze dried to prepare the purifiedbiosurfactant.

The brown precipitate obtained by acid precipitation method isidentified as Brevibacillus agri. Generally, the surfactant produced byBrevibacillus agri is mainly the lipopeptide, which is a kind of chargedbiosurfactant. Therefore the extraction effect is better when using theequal-charge acid precipitation method.

3. Qualitative Analysis By Thin Layer Chromatography(TLC)

The thin-layer plate used in the experiment was silica gel plate, i.e.the adsorbent was silica gel. Therefore the results of thin-layerchromatography were mainly affected by the developing solvent.

(1) Preparation of developing solvent: the developing solvent waschloroform/methanol/water (65/15/2, V/V/V).

(2) It is better to first saturate the space of the chromatographychamber with the vapor of the developing agent. In order to acceleratethe saturation process, the filter paper with developer solvent can besuspended in the chamber.

(3) The spotted thin layer plate was immersed into the developingsolvent with a depth of 0.5-1.0 cm.

(4) When the frontier of the developing solvent moved to about 0.5-1.0cm from the upper edge of the thin layer plate, the development of thedeveloping solvent was stopped. The thin layer plate was quickly takenout and placed flat, and position of the frontier of the solvent wasmarked with a pencil.

(5) Visualization: the visualization reagent was put into the sprayer,and the visualization reagent was uniformly sprayed on the thin layer bywashing ear ball, and then baked in the oven at 110° C. for 10 min.

TLC analysis and staining results showed that staining with ninhydrindirectly failed to visualize. The thin layer plate was put into a sealedflask containing concentrated hydrochloric acid, hydrolyzed in situ at150° C., and then stained with ninhydrin. Red spots were observed,indicating that there was no free amino acids, and free amino acidscould be produced after acid hydrolysis, indicating that the substanceitself did not contain free amino acids, which proved that it was alipopeptide surfactant.

Example 3: Optimization of Lipopeptide-Producing Culture System of B5-2Strain

The culture medium included seed culture medium, fermentation culturemedium and slant culture medium

Seed culture medium (g/L): MgSO_(4 0.2), K₂HPO₄ 1.0, KH₂PO₄ 1.0, NH₄NO₃1.0, yeast extract paste 1.0, CaCl₂ .2H₂O 0.02, FeCl₃ 0.05, distilledwater 1000 mL, pH 7.2;

Culture medium (g/L): MgSO₄ 0.2, K₂HPO₄ 1.0, KH₂PO₄ 1.0, Na₂HPO₄ 4.0,NaCl 10.0, peptone 10.0, crude oil 20.0, molasses 10.0, distilled water1000 mL, pH 7.0;

Slant culture medium (g/L): beef extract 5.0, peptone 10.0, NaCl 5.0,agar 20.0, distilled water 1000 mL, pH 7.0.

1. Optimization of Carbon Source

Nutrients that can be used as carbon source in microbial cell structureor metabolites are all called carbon source. There are a wide range ofcarbon sources that can be used as microbial nutrients, ranging fromsimple inorganic substances (CO₂, carbonate) to complex organic carboncompounds (sugars, sugar derivatives, lipids, alcohols, organic acids,hydrocarbons, aromatic compounds, etc.). However, differentmicroorganisms have different ability to utilize carbon sources. Somecan widely use different types of carbon sources. For example, somespecies of Pseudomonas can use more than 90 kinds of carbon sources.However, the range of carbon sources that some microorganisms can use isvery narrow. For example, Methylosinus trichosporium can only utilizemethane and methanol; some cellulose decomposing bacteria can only usecellulose.

Screening an appropriate carbon source is one of the most importantsteps in the microbial enhanced oil recovery(MEOR). Considering oilfieldapplications and cost requirements, the mixture of heavy wax oil,vegetable oil, crude oil, liquid paraffin, crude oil and molasses isusually used as carbon source.

Based on the quality of carbon source, the dosage of carbon source is2%, the inoculation amount of B5-2 strain is 5%, the culture temperatureis 25° C., the culture time duration is 4 days, and the shaking speed ofshaker is 180 r/min. The result of screening is shown in FIG. 4 .Through the screening of different carbon sources, the result shows thatthe more suitable carbon source for lipopeptide production of B5-2strain is the mixture of crude oil and molasses. Crude oil and molassescan promote the production of lipopeptide biosurfactant better thanother carbon sources.

2. Optimization of Nitrogen Source

Nutrients that constitute the source of nitrogen in microbial cellsubstances or metabolites are all called nitrogen source. Its content inthe dry matter of cells is second only to carbon and oxygen. Nitrogen isan important element of nucleic acid and protein. Therefore, it plays animportant role in the growth and development of microorganisms. Nitrogencan be used by different microorganisms, regardless of molecularnitrogen or complex nitrogen compounds. However, different types ofmicroorganisms can use different nitrogen sources. Nitrogen is animportant component, which is the main elements of living substancessuch as protein and nucleic acid. Nitrogen accounts for 12%-15% of thedry weight of cells. Therefore, similar to carbon source, nitrogensources are also the main nutrient of microorganisms. Yeast extractpaste, peptone, (NH₄)₂HPO₄, sodium nitrate and urea are usually used asnitrogen sources for screening.

Yeast extract paste, peptone, (NH₄)₂HPO₄, sodium nitrate, urea andammonium sulfate were used as nitrogen sources. the amount of nitrogensources was 1%. The amount of carbon source crude oil and molassesmixture was 2%. the inoculation amount of B5-2 strain in seed liquid was5%. The culture temperature was 25° C. The culture time duration was 4days, and the shaking speed was 180 r/min.

Nitrogen source is the basic nutrient source for the synthesis of cellsubstances, and its type and concentration determine the growth ofbacteria, which affects the production of lipopeptide biosurfactant.After screening, it is found that, as shown in FIG. 5 , in the aspect oflipopeptide production, inorganic nitrogen is better than organicnitrogen, and sodium nitrate is the best inorganic nitrogen.

3. Optimization of Culture Medium By Orthogonal Experiment

Single factor experiment was used to optimize the carbon source,nitrogen source and mineralization of fermentation culture medium, aswell as the seed age, inoculation amount, temperature, oxygen demand andpH in the culture process. In addition, other components in the culturemedium were also optimized and adjusted. L₁₆ (4⁵) orthogonal experimentwas used to reduce the number of experiments, as shown in Table 3 andTable 4.

TABLE 3 Factors and levels of orthogonal experiment Factors Crude oil +Sodium Sodium Culture molasses nitrate chloride temperature Levels g/Lg/L g/L ° C. 1 3 5 5 25 2 6 10 10 29 3 9 15 15 33 4 12 20 20 37

TABLE 4 Results of L₁₆ (4⁵) orthogonal experiment A crude E surfaceoil + B Sodium C Sodium D Culture tension molasses nitrate chloridetemperature m Number g/L g/L g/L ° C. Nm⁻¹ 1 3 5 5 25 36.85 2 3 10 10 2934.94 3 3 15 15 33 37.54 4 3 20 20 37 40.42 5 6 5 10 33 38.47 6 6 10 537 39.41 7 6 15 20 25 30.83 8 6 20 15 29 31.42 9 9 5 15 37 41.52 10 9 1020 33 36.88 11 9 15 5 29 35.48 12 9 20 10 25 29.45 13 12 5 20 29 34.5814 12 10 15 25 31.29 15 12 15 10 37 37.53 16 12 20 5 33 33.51 Mean 137.438 37.855 36.312 32.105 Mean 2 35.032 35.630 35.097 34.105 Mean 335.832 35.345 35.443 36.600 Mean 4 34.227 33.700 35.677 39.720 Range3.211 4.155 1.215 7.615

The intuitive analysis method is used for the results of the orthogonalexperiment. The results of the intuitive analysis are shown in Table 3and Table 4. From the calculation results in Table 4, FIG. 4 and FIG. 5, it can be seen that A₄B₄C₂D₁ is the optimal combination. i.e. theconcentration of carbon source is 12.0 g/L, the concentration of sodiumnitrate is 20.0 g/L, the concentration of sodium chloride is 10.0 g/L,and the culture temperature is 25° C., the gradient analysis result isR_(D)>R_(B)>R_(A)>R_(C). i.e. the culture temperature has the greatestinfluence on the lipopeptide production.

Example 4: The Effect On Crude Oil (Heavy Oil)

1. Emulsifying Property

Emulsification was a phenomenon in which water or organic solvents aredispersed in the organic or aqueous phase in the form of tiny droplets.i.e. when a liquid phase is dispersed in droplets in another continuousliquid phase, an emulsion is formed. 5 mL of liquid paraffin and 5 mL ofsurfactant solution were added into a test tube, and the test tube wasoscillated with a vortex oscillator for 1 min and then kept still. Thevolume of the aqueous phase, emulsified phase and oil phase weremeasured at different times. The emulsifying capacity is expressed bythe ratio of the volume of emulsified phase to the total volume.

The results of the emulsification stability of lipopeptide biosurfactantproduced by B5-2 strain to kerosene and liquid paraffin are shown inFIG. 6 . It can be seen from FIG. 6 that lipopeptide biosurfactant hasgood emulsifying capacity. After 96 hours, the emulsifying capacity oflipopeptide biosurfactant produced by B5-2 strain on kerosene oil andwater can still be maintained above 72%, so it is a good emulsifier.This is because the biosurfactant is a lipopeptide mixture containingmultiple components, with diverse structures and strong affinity to theoil/water interface, which stabilizes the emulsified phase, which provesthat the lipopeptide has a good application prospect.

2. Results of Oil Spreading Activity

Water was added to a culture disk, and 200 μL n-dodecane was dripped inthe culture disk. After the oil film was formed, 10 μL fermentationliquid was dripped into the center of the oil film. The diameter andstability of the clear zone was observed. Experiment for three parallelsamples was conducted to give a deviation within 5%, and the averagevalue was taken as the clear zone diameter of the sample.

The lipopeptide biosurfactant produced by B5-2 strain has a diameter of6.6 cm for n-dodecane, indicating that it has good oil drainageperformance.

3. Results of Test For Electricity Charging of Biosurfactant

(1) Methylene blue-chloroform method was used to determined anionicsurfactant.

5 mL sample was added into 25 mL test tube, then 10 mL methylene bluesolution and 5 mL chloroform were added. The test tube was shaken forseveral minutes. If the solution contains anionic surfactants, then thechloroform layer turns blue.

(2) Bromophenol blue method was used to determined cationic surfactant.

The pH of the sample solution was adjusted to 7.0, 5 mL of the samplesolution was added into the 25 mL test tube, and then 2-5 drops of theprepared bromophenol blue solution were added into the test tube. If thesolution turns dark blue, it contains cationic surfactants.

The results of the determination method of anionic surfactant (Methyleneblue-chloroform method) and cationic surfactant (bromophenol bluemethod) showed that the chloroform layer turned blue, indicating thatthe biosurfactant in the sample was an anionic surfactant.

4. Changes of Surface Tension, PH and Viscosity Before and After theAction of B5-2 Strain

Surface tension is an important parameter of liquid, which is usuallyused to reflect the surface properties of liquid, i.e. the amount ofsurfactants in liquid. The lower the surface tension is, the higher thecontent of surfactants in the liquid. In the process of microbialenhanced oil recovery, some microbial metabolites contain certainsurfactants, which are generally called biosurfactant. Biosurfactant canreduce the interfacial tension of water/oil/rock, emulsify crude oil,and change the surface wettability of reservoir pores. However, it isdifficult to directly measure the content of biosurfactant in microbialfluid. The surface tension is often used to evaluate the activity of themicrobial liquid. Microbial metabolites such as acid and surfactantproduction are the important mechanism of microbial enhanced oilrecovery. The ability of reducing surface tension and pH change are thebasic indexes to evaluate the performance of bacteria. The ability ofproducing surfactant can reduce the interfacial tension of oil andwater, and reduce the capillary force of enhanced oil, so as to improveoil recovery. The ability of microorganism to reduce the oil-waterinterface can be used to determine the ability of the strains to produceactive agents.

(1) Study On Surface Tension and PH

200 mL fermentation culture medium was put into 500 mL triangular flaskand cultured on a rotary shaker (180 r/min) at the actual temperature ofthe oilfield for 4 days. The fermentation broth was centrifuged toremove the bacteria, and then the supernatant was filtered with filterpaper to remove the residual oil in the supernatant as much as possible.

The surface tension was measured by DCA322 contact angle analyzer fromCAHN company of US. The hanging plate method was used to measure thesurface tension of microbial liquid. The whole measuring process of theinstrument was automatically completed under the control of a computerprogram.

In the test, the nutrition solution was prepared by using distilledwater and Distilled water was used as the reference sample. Thedetermined surface tension of blank culture medium was 64.38 mN/m.

B5-2 strain solution was cultured under different temperature andmineralization degree for 4 days, and then oil and water were separated.The pH and surface tension of bacteria solution were measured. Theexperimental data was shown in Table 5. The results show that:

1) B5-2 strain reduces the surface tension of oil samples from three oilwells in a western oilfield, but the reduction degree of the surfacetension of different crude oils by B5-2 strain is different. The surfacetension of TH191 crude oil, TH291 crude oil and TH222 crude oil aredecreased by 50.26%, 45.46% and 47.91%, respectively.

2) The pH decreased from 7.2 to acid, indicating that acid is producedin the degradation process.

TABLE 5 Changes of surface tension and pH of crude oil treated by B5-2strain Reduction rate Sample Temperature Surface tension (%) pH TH19125° C. 32.02 50.26 6.5 TH291 25° C. 35.11 45.46 6.6 TH222 25° C. 33.5347.91 6.3

(2) Change of Crude Oil Viscosity

In oil recovery engineering, crude oil viscosity is a key factoraffecting crude oil production. The lower the oil viscosity and thebetter the fluidity, the easier it is to be recovered out of the ground.Therefore, during the development of oil recovery technology, variousmethods of reducing the crude oil viscosity have been studied. Manyprevious research results show that some microorganisms can reduce thecrude oil viscosity after interacting with crude oil. There are threereasons. First, the degradation of crude oil by microorganisms destroysthe network structure formed by colloids and asphaltenes in crude oil asparticles, i.e. it reduces the relative content of heavy components incrude oil and improves the fluidity of crude oil. Second, microorganismsproduce some organic solvents in the process of reproduction andmetabolism to dilute crude oil. The third reason may be that thesurfactant produced by microbial metabolism has an emulsifying effect oncrude oil to form oil-in-water emulsion, thus reducing the viscosity ofcrude oil. The ability of microorganisms to reduce the viscosity is alsoconsidered an important parameter when screening and evaluatingmicrobial strains for oil recovery.

In the experiment, the viscosity was measured by BROOK FILELD II tumblefluidimeter imported from the US. 200 mL fermentation culture medium wasfilled in 500 mL triangular flask and cultured on a rotary shaker (180r/min) at the actual temperature of the oilfield for 4 days. After 4days, the bacteria solution was separated from the oil, centrifuged by ahigh-speed centrifuge at 9000 rpm for 10 minutes to dehydrate the oil.The dehydrated oil sample was poured into the fluidimeter. Thetemperature of the circulating water bath of the fluidimeter is set to65° C., install U2 rotor on the fluidimeter. The viscosity of the oilsample was measured at the speed of 1.5 RPM after the sample temperaturein the fluidimeter was stable. The viscosity reduction ability ofbacteria solutions through the viscosity change before and after theaction of oil and bacteria solution were compared.Viscosity reduction rate=(η_(before)-η_(after))/η_(before)*100%

η_(before) is the viscosity of crude oil before microbial action, andη_(after) is the viscosity of crude oil after microbial action.

The results of crude oil viscosity reduction by indigenous bacteria B5-2are shown in Table 6.

TABLE 6 Changes of crude oil viscosity after the action of B5-2 strainviscosity Before viscosity After viscosity reduction Sample reduction(mPa · s) reduction (mPa · s) rate (%) TH191 46.9 31.3 33.3 TH291 51.336.5 28.8 TH222 52.2 35.2 32.6

The results of viscosity reduction experiment in Table 6 show that theB5-2 strain has good viscosity reduction effect on the three kinds ofcrude oil. The viscosity reduction rates of B5-2 on TH191 crude oil,TH291 crude oil and TH222 crude oil are 33.3%, 28.8% and 32.6%,respectively.

Example 5: Changes in Components of Crude Oil Before and After BacterialDegradation Of B5-2

1. Component Analysis of Crude Oil

Changes in the components before and after the action of the indigenousbacteria on the crude oil of Xinjiang Oilfield were evaluates indoor.The four components are saturated hydrocarbon, aromatic hydrocarbon, gumasphaltene and non-hydrocarbon.

After 7 days of cultivation at 25° C. and 180 r/min in air shaker, thecrude oil was degraded to a certain extent and emulsified. The crude oilwas collected in the sample flask, centrifuged on the high-speedcentrifuge (8000 r/min, centrifugation time is 1 min), and the water atthe bottom of the sample flask was removed with 5 mL syringe. The sampleflask was put in a refrigerator at 4° C. Then four component analysisand mass spectrometry analysis were performed. The content of eachcomponent of crude oil changes, as shown in Table 7 and FIG. 7 .

TABLE 7 Relative content of crude oil components before and after theaction of B5-2 strain Saturated Aromatic Non- hydrocarbon hydrocarbonhydrocarbon Asphaltene Items (%) (%) (%) (%) Before 41.38 26.29 22.412.16 degradation After 56.85 16.33 14.29 6.12 degradation S-4-OK

The results of the B5-2 strain degradation test of crude oil in thewestern oilfield show that the relative content of saturatedhydrocarbon, aromatic hydrocarbon, non-hydrocarbon and asphaltene haschanged significantly. The relative content of saturated hydrocarbon andasphaltene increases, while the relative content of aromatic hydrocarbonand non-hydrocarbon decreases in varying degrees, which indicates thatmicroorganisms preferentially degrade high carbon chain components,resulting in the increase of relative content of saturated hydrocarbon,and degradation of aromatic hydrocarbon and non-hydrocarbon componentsby B5-2 strain, and increasing the relative content of asphaltenes.

2. Results of Chromatography-Mass Spectrometry

Executive standard: GB/T 18606-2001 The Standard Test Method forBiomarker in Sediment and Crude Oil By GC-MS

Instrumentation: Agilent 7890-5975c chromatography-mass spectrometer

Test conditions: chromatography; carrier gas: 99.999% helium; injectionport temperature: 300° C.; transmission line temperature: 300° C.;chromatographic column: HP-5MS elastic quartz capillary column (60m×0.25 mm×0.25 m); column temperature: initial temperature: 50° C. for 1min; 20° C./min to 120° C., 4° C./min to 250° C., then 3° C./min to 310°C., keep for 30 mins; carrier gas flow rate: 1 mL/min; mass spectrometryEI source, absolute voltage: 1047 V; full scan.

As shown in FIG. 8 and FIG. 9 , the ratio of pristane (Pr) and phytane(Ph), n-nonadecane and n-eicosane changes to a certain extent, therelative content of C27 and C28 decreases, and the proportion of alkaneswith carbon number less than 16 increases after the action of B5-2strain, indicating that the high carbon chain component is transformedinto low carbon chain component by the indigenous bacteria, thus thelight components increase. GC/MS analysis showed that B5-2 strain hasnot degrade low-carbon number saturated hydrocarbons significantly, andmainly degrades some high carbon alkanes.

As shown in FIG. 10 and FIG. 11 , aromatic hydrocarbons are obviouslydegraded, especially the aromatic hydrocarbons below C20.Trimethylnaphthalenes are significantly degraded, phenanthrenes aresignificantly degraded, and the proportion ofdimethylnaphthalene/trimethylnaphthalene and phenanthrene issignificantly changed. GC/MS analysis results show that B5-2 strainpreferentially degrade naphthalene and phenanthrene, and mainly degradearomatic hydrocarbons below C20. The results show that B5-2 strain caneffectively degrade the aromatic hydrocarbons below C20 and make thecomponents of crude oil change obviously.

3. Changes of Polar Heteroatom Compounds

The composition changes of TH191 heavy oil before and after the actionof B5-2 strain were analyzed by linear ion trap fourier-transform ioncyclotron resonance mass spectrometry (FT-ICR). After microbialdegradation, the content of N compounds in crude oil decreased fromabout 70% to less than 20%, and the content of O₂ compounds decreasedfrom more than 20% to less than 5%. The proportions of NO and NO₂decreases in varying degrees. NO₃ and NS compounds may be decomposed bymicroorganisms, while the content of O₃ compounds increasessignificantly, with the proportion being more than 70%.

Relative abundance is defined as the intensity of each peak in the massspectrum divided by the sum of the intensities of all identified peaks(except for isotopic peaks). Even if the relative abundance of a givenclass in two samples is the same, its absolute abundance is notnecessarily the same, because it depends on the abundance of otherclasses. The relative abundances of crude oil TH191 and heteroatomsafter the action of B5-2 strain are shown in FIG. 12 and FIG. 13 . InFIG. 12 , N compounds account for the majority of heteroatoms, more than70%, and other types of heteroatoms accounted for a relatively lowproportion: the content of O₂ is the second, more than 20%. The contentof NO, NO₂, O are less than 5%, with trace amounts of NO3 and NS. whichcan be ignored. As shown in FIG. 12 , the content of N compounds aftermicrobial degradation decreases significantly, less than 20%. Theproportion of NO and NO₂ decreases. After microbial degradation, NO₃ andNS compounds are decomposes by microorganisms, which are not shown inFIG. 13 . However, O₃ increases significantly, accounting for more than70%, which may be due to the formation of a large amount ofoxygen-containing acids. It shows that after the action of B5-2 strain,the neutral nitrogen heteroatom compounds of the polar compounds in theoil-water interface are degraded obviously. As a result, the formationof highly hydrophilic acidic compounds, resulting in the change of thehydrophobicity and lipophilicity of the interfacial substances on thecrude oil, i.e. the lipophilicity of crude oil-water interfacial activesubstances is reduced. The hydrophilicity of crude oil is enhanced,which eventually lead to the reversing of the crude oil emulsion andforming the oil-in-water emulsion. In addition, the viscosity is greatlyreduced, the fluidity and the oil recovery is enhanced.

Example 6: Simulation Test For Oil Displacement

. Preparation of Bacteria Solution For Oil Displacement

150 mL surfactant bacteria fermentation culture medium and 3 L inorganicsalt culture medium were prepared. The medium was autoclaved at 121° C.for 20 min. B5-2 strain was activated and inoculated to the fermentationculture medium, and cultured at 25° C. and 180r/min for 24 h. Thefermentation broth was added to 3L enhancing oil medium at 10%, andcultured at 25° C. and 180 r/min for 4 days. The fermentation broth wastaken out and put into a 4° C. refrigerator.

2. Physical Simulation Test For Core Flooding

The process of oil displacement was connected, which process wascomposed of microinjection pump (gas cylinder for pressure source ofconstant pressure displacement), high pressure vessel, core model andoil-water separation metering tube. The above four parts were connectedby pipelines and valves. The pressure gauge was installed as required todisplay the pressure values. The parts that need to maintain theexperimental temperature were installed in the incubator.

Two artificial rock cores of TH121 and TH122 with gas permeability of100-200md were selected to inject B5-2 strain bacteria solution 2PV, andshut in the well for 4 days, and the oil displacement effect wasmeasured.

(1) Saturation Of Core With Water

The artificial core with gas permeability of about 200md was weighed thedry weight and then put into a wide mouth bottle. The wide mouth bottlewas vacuumed with vacuum pump for three hours, and then the simulatedformation water was added to make the core completely immersed in water.The vacuum pump was turned on until the vacuum gauge read zero, and thenthe artificial core was taken out, and the wet weight was weighed to getthe of pore volume the core.

(2) Saturation Of Core With Oil

The core was put into a core holder, and the core holder was put into a40° C. incubator. The dehydrated crude oil was added into a steelvessel. The pipeline was connected. The constant-flux pump was turned onto press the crude oil into the core. When 3-4 mL crude oil flowed outof the outlet end of the core holder, the saturated oil was over. Thecrude oil saturation was obtained based on the volume of the drivenwater.

(3) Water Flooding

The simulated formation water was put into the steel container. Thepipeline for oil displacement was connected. The constant-flux pump wasturned on for oil displacement. 5 mL test tube was used to receive thecrude oil at the export of the core holder, and counting was made every10 minutes until the water content reached 95%.

(4) Oil Displacement By Microorganism

The B5-2 strain bacteria solution was put into a steel container, thepipeline was connected for oil displacement. The amount of crude oilexpelled was recorded. The constant-flux pump was closed after reachingthe specified PV. The B5-2 strain bacteria solution was shut in the wellat 50° C. for 4 days.

(5) Follow Up Water Flooding

After the specified well-shut time, follow-up water flooding wasperformed on the core, the amount of crude oil driven was recorded, andthe value of the enhanced crude oil recovery factor was calculated.

The cores used for biosurfactant producing B5-2 strain were TH121 andTH122. The physical properties of two homogeneous artificial cores areshown in Table 8. When the two cores were water flooded to 95% of watercontent, the biosurfactant produced by B5-2 strain bacteria solution wasinjected, and shut in the well for 4 days. The subsequent water floodingwas performed to obtain the recovery ratio. The results are shown inTable 8.

TABLE 8 physical properties of homogeneous cores Length Pore volumePorosity Permeability Diameter mm mm mL % 10⁻³ μm² Blank 25 71.5 9.3325.12 167.54 TH121 25 70.3 8.79 25.23 161.32 TH122 25 69.5 8.68 25.59158.33

Table 8 shows the physical properties of TH121 and TH122. The physicalparameters of the two cores are roughly the same. The gas permeabilityis about 160 md, the pore volume is about 25%, and the oil saturation isabout 82%. According to the B5-2 strain enhancing oil experimental data,for TH121 and TH122 cores, the increase in oil recovery rate after theaction of 120mg/L B5-2 strain bacterial solution is 10-12%,respectively. The enhancing oil effect of the B5-2 strain is obvious.

The present disclosure is not limited by the above-mentioned examples.The above-mentioned examples and the description only illustrate theprinciple of the present disclosure. On the premise of not departingfrom the spirit and scope of the present disclosure, there will bevarious changes and improvements. These changes and improvements fallinto the scope of the present disclosure claimed. The scope of thepresent disclosure is defined by the attached claims.

What is claimed is:
 1. A method for preparing biosurfactant, wherein themethod comprises fermenting a Brevibacillus agri strain in a nutrientmedium to produce a lipopeptide biosurfactant, wherein the Brevibacillusagri strain is Brevibacillus agri deposited with the China GeneralMicrobiological Culture Collection Center (CGMCC) with CGMCC depositnumber 9983, wherein the nutrient medium comprises: MgSO₄ 0.2 g/L,K₂HPO₄ 1.0 g/L, KH₂PO₄ 1.0 g/L, Na₂HPO₄ 4.0 g/L, NaCl 10.0 g/L, NaNO₃10.0 g/L, crude oil 10.0 g/L and molasses 2-3 g/L, pH is 7.0, and afermentation temperature is 25-60° C.
 2. The method according to claim1, wherein the nutrient medium further comprises 1-5 mL of a solution oftrace elements and 1-10 mL of a solution of a vitamin complex.
 3. Themethod according to claim 1, further comprising removing theBrevibacillus agri strain after fermentation.
 4. The method according toclaim 1, further comprising obtaining the lipopeptide biosurfactant. 5.The method according to claim 1, wherein the lipopeptide biosurfactantis a metabolite of the Brevibacillus agri strain.
 6. The methodaccording to claim 1, wherein the Brevibacillus agri strain is a liquidbacterial preparation.
 7. The method according to claim 6, wherein thenutrient medium further comprises 1-5 mL of a solution of trace elementsand 1-10 mL of a solution of a vitamin complex.
 8. The method accordingto claim 6, further comprising removing the Brevibacillus agri strainafter fermentation.
 9. The method according to claim 4, wherein thenutrient medium further comprises 1-5 mL of a solution of trace elementsand 1-10 mL of a solution of a vitamin complex.
 10. The method accordingto claim 4, further comprising removing the Brevibacillus agri strainafter fermentation.